Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Transport by vesicles of glycine- and taurine-conjugated bile salts and taurolithocholate 3-sulfate: A comparison of human BSEP with rat Bsep
Introduction
Bile formation is one of important functions of the liver. Vectorial transport of bile salts from the sinusoidal space to the canaliculus via hepatocytes provides an osmotic driving force for bile formation [1], [2]. Transport of bile salts across the sinusoidal membrane is mediated at least in part by both Na+-taurocholate co-transporting polypeptide (human NTCP/SLC10A1 and rat Ntcp/Slc10a1) [3], [4] and Na+-independent organic anion transporting polypeptides (human OATP/SLCO and rat Oatp/Slco) [1], [2]. After reaching the canalicular membrane, monovalent taurine- and glycine-conjugated bile salts are secreted into bile by the bile salt export pump (human BSEP/ABCB11 and rat Bsep/Abcb11) [5], [6], [7], [8], [9], [10], whereas sulfated bile salt amidates and bile salt ethereal glucuronides (divalent) are excreted by the multidrug resistance associated protein 2 (human MRP2/ABCC2 and rat Mrp2/Abcc2) [11], [12], [13].
The transport properties of hBSEP/rBsep and hMRP2/rMrp2 have been clarified recently. The function of hBSEP/rBsep has been characterized by examining the ATP-dependent transport of taurine- and glycine-conjugated bile salts in isolated bile canalicular membrane vesicles (CMVs) and/or membrane vesicles isolated from hBSEP/rBsep-expressing cells [5], [6], [7], [8]. For hMRP2/rMrp2, the transport characteristics have been mainly investigated by comparing transport across the bile canalicular membrane in normal rats with that in Mrp2-deficient rats [8], [14], [15], [16]. Mutations in BSEP gene are now known to be the cause of progressive familial intrahepatic cholestasis type 2 (PFIC2) [17], [18], [19], [20], [21], whereas mutations in MRP2 are the cause of Dubin–Johnson syndrome (DJS) [22], [23], [24].
Major bile salts in mammals, i.e., ionized forms of C24-bile acids, are synthesized from cholesterol in the liver and then conjugated (N-acylamidated) with glycine or taurine. In health, sulfation of primary bile salts does not occur in most mammals, but in cholestasis at least in some species, bile salts are extensively sulfated and eliminated into urine. The biliary bile salt composition in the human significantly differs from that in the rat. In humans, the majority of bile salts are conjugated with glycine, whereas in rats, most bile salts are conjugated with taurine. Human bile, but not rat bile, contains sulfated and unsulfated amidates of lithocholate (LCA) [25], [26].
Two groups have attempted to relate the transport properties of hBSEP to biliary bile salt composition in humans as compared to that in rats [5], [7]. Noe et al. concluded that the transport property of hBSEP/rBsep does not explain the differences in steady state biliary bile salt composition between the two species, because of the similarity of Km value and intrinsic clearance value for TC, GC, TCDC and TUDC [7]. On the other hand, Byrne et al. concluded that the transport properties of hBSEP/rBsep do in fact correlate with the different bile salt pools in human and rat due to the difference of the relative affinities for TC, GC, TCDC and GCDC between hBSEP and rBsep [5]. In the present study, we have characterized the transport function of hBSEP and rBsep for twelve physiologic bile salts including several kinds of bile salts untested in the previous reports, such as taurolithocholate 3-sulfate. The transport function was determined using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP and rBsep cDNA. We were able to confirm that the transport properties of hBSEP/rBsep reflect the difference in bile salt composition between human and rat. Kinetic analyses to hBSEP and hMRP2 were also performed for [3H]TLC-S, since the initial uptake study demonstrated that this sulfated bile salt is good substrate for hBSEP, not good substrate for rBsep.
Section snippets
Materials and cell culture
[3H]cholate (CA) (24.5 Ci/mmol), [3H]taurocholate (TC) (2 Ci/mmol), [14C]chenodeoxycholate (CDCA) (48.6 mCi/mmol) and [2-3H]taurine (30.3 Ci/mmol) were purchased from NEN Life Sciences Products (Boston, MA). [14C]glycocholate (GC) (57.3 mCi/mmol) and [14C]lithocholate (LCA) (57.3 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). [3H]ursodeoxycholate (UDCA) (20 Ci/mmol), [3H]tauroursodeoxycholate (TUDC) (10 Ci/mmol), [3H]glycoursodeoxycholate (GUDC) (11
Uptake of [3H]TC into membrane vesicles
The expression of hBSEP or rBsep in the membrane vesicles prepared from the transfected HEK293 cells was confirmed by Western blot analysis (Fig. 1). As shown in Fig. 1, hBSEP and rBsep were detected as an approximately 170 kDa form in the fraction of membrane vesicles prepared from hBSEP- and rBsep-transfected HEK 293 cells. No expression of hBSEP or rBsep was detected in the control membrane vesicles prepared from GFP-transfected HEK293 cells.
The time-profiles for the uptake of [3H]TC into
Discussion
In the present study, we compared the transport properties of hBSEP and rBsep using membrane vesicles from HEK293 cells infected with adenoviruses containing these cDNAs. Our aim was to examine the correlation between the function of hBSEP/rBsep and the biliary bile salt composition.
Initially, transport studies were performed by measuring the ATP-dependent uptake of [3H]TC into the hBSEP- and rBsep-expressing membrane vesicles to confirm the transport function of hBSEP and rBsep. The Km values
Acknowledgements
We thank Mr. Masakazu Hirouchi for construction of hMRP2 expression system, Dr. Hidetaka Akita and Ms. Sachiko Mita for construction of rBsep expression system. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas Epithelial Vectorial Transport 12144201 and Grant-in-Aid for Center of Excellence (COE) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Work in the laboratory of Alan F. Hofmann is supported by NIH Grant DK 64891.
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